Antigen protein and nucleic acid coding for said protein

ABSTRACT

To provide a novel antigenic protein and a nucleic acid encoding the antigenic protein, which are useful for prophylaxis, treatment and diagnosis of diseases caused by fungi including  Candida albicans . An antigenic protein characterized in that the antigenic protein is recognized by antiserum derived from a mammal having  Candida albicans -infection resistance; and a nucleic acid encoding an antigenic protein which is recognized by antiserum derived from a mammal having  Candida albicans -infection resistance.

This application is a Divisional of co-pending application Ser. No.09/509,744 filed on Mar. 31, 2000 and for which priority is claimedunder 35 U.S.C. § 120. Application Ser. No. 09/509,744 is the nationalphase of PCT International Application No. PCT/JP98/04326 filed on Sep.28, 1998 under 35 U.S.C. § 371. The entire contents of each of theabove-identified applications are hereby incorporated by reference. Thisapplication also claims priority of Application No. 9-269087 filed inJapan on Oct. 1, 1977 under 35 U.S.C. § 119.

TECHNICAL FIELD

The present invention relates to Candida albicans antigenic protein, anda nucleic acid encoding thereof, which are useful for prophylaxis,treatment and diagnosis of fungal infectious disease or allergosis. Thepresent invention further relates to a vector comprising the nucleicacid, and a transformant resulting from transformation with the vector.The present invention further relates to a method for producing theantigenic protein of the present invention, wherein the method comprisesculturing the above transformant. The present invention further relatesto a pharmaceutical composition or diagnostic composition, eachcomprising the antigenic protein or nucleic acid of the presentinvention.

BACKGROUND ART

Although Candida albicans normally colonizes in the human body, normalindividuals are usually resistant to infection therewith and seldomsuffer from systemic infectious disease caused thereby. Even in normalindividuals, however, this fungus can cause local infectious disease; infemales, particularly in pregnant women, it can cause vaginalcandidiasis. On the other hand, in humans rendered immunocompromised asa result of administration of an anticancer agent for treatment of acancer, such as leukemia, administration of an immunosuppressant fororgan transplantation, infection with AIDS, or the like, this fungus cancause systemic infectious diseases affecting various internal organs.Furthermore, this fungus represents the most common fungal allergen as acause of allergosis. In addition, most humans are immunologicallysensitized with the normally colonizing fungus Candida albicans, possessantibodies against its components, and have acquired cellular immunitythereto.

Many of the Candida albicans antigens are cell wall components on thecell surface, mainly polysaccharides, such as mannan and glucan, mannanand its conjugate with protein being particularly predominant. Inaddition, in sera from patients with Candida albicans infection or thosewith allergy, antibodies against the intracellular components enolase,HSP90 (heat shock protein 90), phosphoglycerate kinase, or alcoholdehydrogenase are detected, in addition to the antibodies against theaforementioned cell wall components.

Fungi of the genus Candida other than Candida albicans, for instance, C.tropicalis and C. glabrata, cause infectious disease mainly inimmunocompromised hosts. Fungi other than those of the genus Candida,for instance, fungi of the genera Aspergillus, Penicillium, andAlternaria, also occur widely in our environment and are very close tomammals, including humans. Although these fungi are yeast-like ormycelial in the cellular morphology to grow, their cellular structuresare basically similar to each other, all having a surface layersurrounded by a thick cell wall. Although the chemical structure of thecell wall varies to some extent depending on kind of fungus, the cellwall components mainly comprise polysaccharides, such as mannan, glucan,and chitin. In addition, the proteins and other components in the cellmembrane and cytoplasm surrounded by the cell wall are also basicallysimilar to each other. Many of these fungi also cause infectious diseasein immunocompromised hosts, or serve as causes of allergy.

As pathogenic factors and allergens in fungus-involved disease, someantigen molecules derived from fungus have been isolated and identified.Isolation and identification of these antigen molecules are usuallyachieved using sera from fungus-sensitized mammals; the most common ofsuch antigen molecules that have been identified so far are the cellwall components surrounding the fungal surface layer. In addition to theabove components, a number of antigen molecules have been isolated andidentified, and are under investigation for the purposes of diagnosisand therapy. It should be noted, however, that not all antigens reactiveto antibodies retained in fungus-sensitized state act effectively ininfectious disease or allergosis.

An object of the present invention is to provide a novel antigenicprotein useful in the treatment and diagnosis of diseases caused byfungi including Candida albicans. Another object of the presentinvention is to provide a nucleic acid encoding the antigenic protein.Still another object of the present invention is to provide a vectorcomprising the nucleic acid, and a transformant resulting fromtransformation with the vector. Still another object of the presentinvention is to provide a method for producing the antigenic protein ofthe present invention, wherein the method comprises culturing thetransformant. A further object of the present invention is to provide apharmaceutical composition and diagnostic composition, each comprisingthe antigenic protein or nucleic acid of the present invention.

DISCLOSURE OF INVENTION

The present inventors have investigated about a method for conferringCandida albicans-infection resistance to mammals, such as mice and rats,which are sensitive to Candida albicans infection. As a result, thepresent inventors have clarified that such mammals can acquire strongresistance to infection by immunizing the mammals with Candida albicanscells as the antigen in mixture with an adjuvant of incomplete Freund'sadjuvant. The present inventors have also clarified that CD4-positiveT-cells play a key role in the infection resistance. Furthermore,antisera derived from the above immunologically sensitized mammalsacquiring the infection resistance have unexpectedly lower antibodytiters against the cell wall-derived components in the fungal cellsurface layer, and higher antibody titers against the components derivedfrom protoplast cells of fungi deprived of cell wall, in comparisonwith, for example, Factor Serum No. 1 (manufactured by IATRONLABORATORIES, INC.), a commercially available anti-Candida serum.

Having assumed that the serum from an immunologically sensitized mammalshowing the above infection resistance is richer in antibodies actingeffectively against fungal infectious disease, and remarked on theantibodies contained in the serum, the present inventors have screenedfor antigenic proteins recognized by the antisera. Concretely, anexpression library was prepared on the basis of Candida albicans cDNAand screened by immunoscreening for such antigenic proteins. As aresult, the present inventors have found eight kinds of antigenicproteins as recombinant Escherichia coli proteins, isolated their DNA,determined the nucleotide sequences, and at the same time determined theamino acid sequences from the nucleotide sequences. The presentinvention has been accomplished thereby.

Concretely, the gist of the present invention relates to:

-   [1] an antigenic protein characterized in that the antigenic protein    is recognized by antiserum derived from a mammal having Candida    albicans-infection resistance;-   [2] an antigenic protein immunologically equivalent to the antigenic    protein according to item [1] above;-   [3] a nucleic acid encoding an antigenic protein which is recognized    by antiserum derived from a mammal having Candida albicans-infection    resistance;-   [4] a vector comprising the nucleic acid according to item [3]    above;-   [5] a transformant resulting from transformation with the vector    according to item [4] above;-   [6] a method for producing an antigenic protein which is recognized    by antiserum derived from a mammal having Candida albicans-infection    resistance, characterized in that the method comprises culturing the    transformant according to item [5] above under conditions capable of    expressing an antigenic protein encoded by the nucleic acid    according to item [3] above;-   [7] a pharmaceutical composition characterized in that the    pharmaceutical composition comprises the antigenic protein according    to item [1] or [2] above, or an antigenic protein obtainable by the    method according to item [6] above;-   [8] a diagnostic composition characterized in that the diagnostic    composition comprises the antigenic protein according to item [1] or    [2] above, or an antigenic protein obtainable by the method    according to item [6] above;-   [9] a pharmaceutical composition characterized in that the    pharmaceutical composition comprises the nucleic acid according to    item [3] above;-   [10] a diagnostic composition characterized in that the diagnostic    composition comprises the nucleic acid according to item [3] above.-   [11] an antibody capable of specifically binding to the antigenic    protein according to item [1] or [2] above, or a fragment thereof;    and-   [12] a nucleic acid capable of specifically binding to the nucleic    acid according to item [3] above.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 shows analytical results of SDS-polyacrylamide gelelectrophoresis of recombinant proteins each derived from lysyl tRNAsynthethase homologue genes and TPI (triose phosphate isomerase)homologue genes of C. albicans.

The sample of lane 1 in FIG. 1 is a polypeptide obtained before theinduction of expression of the transformant in which the lysyl-tRNAsynthase homologue gene (before addition of IPTG) was transduced; andthe sample of lane 2 is a polypeptide obtained after the induction(after addition of IPTG). The sample of lane 3 comprises the polypeptideobtained before the induction of expression of the transformant in whichthe TPI homologue gene was transduced, and the sample of lane 4 is thepolypeptide obtained after the induction.

BEST MODE FOR CARRYING OUT THE INVENTION

1. Antigenic Proteins of Present Invention

The antigenic protein of the present invention is a protein recognizedby antiserum derived from a mammal having Candida albicans-infectionresistance.

In the present specification, the term “mammal having Candidaalbicans-infection resistance” refers to a mammal allowed to acquireinfection resistance by immunization with antigens derived from Candidaalbicans, the mammal being alive even when Candida albicans cells at alethal number before immunization are administered (dead cell countswithin 10 days after intravenous infection).

Such a mammal can, for example, be obtained in the manner describedbelow.

At least two subcutaneous or intraperitoneal administrations are givenat an interval of 1 to 2 weeks, of a mixture of live Candida albicanscells as the antigen and incomplete Freund's adjuvant (IFA) for stronginduction of cellular immunity to an animal sensitive to Candidaalbicans infection, including, for instance, a mouse such as C57BL/6,BALB/c, or DBA/2, a rat such as Sprague-Dawley or Wistar, etc., therebyimmunizing the animal. The animal which has acquired infectionresistance is used as a mammal having Candida albicans-infectionresistance.

In the present specification, the term “antiserum derived from a mammalhaving Candida albicans-infection resistance” refers to an antiserumobtained from a mammal having Candida albicans-infection resistance.

As the above antiserum, there can be used, for example, those obtainedby collecting blood from a mammal having Candida albicans-infectionresistance at 1 to 2 weeks after final immunization. Such antiserum isan antiserum obtained after 1 week to 1 month following at least twoimmunizations at an interval of about 1 week, wherein preferably amouse, more preferably a 5 weeks old or more, such as C57BL/6, BALB/c orDBA/2, is used; 1×10⁵ to 1×10⁸ live cells are mixed with IFA, to give amixture; and thereafter the above mouse is immunized with the resultingmixture. The antiserum used in the present invention shows high antibodytiters against the cell membrane components and intracellular moleculesof fungal cells. In contrast, the anti-Candida antisera generally usedare not derived from mammals having infection resistance, and possesshigh antibody titers against the surface cell wall components, forinstance, mannan and mannoprotein, which are different from theantiserum in the present invention.

The Candida albicans cells usable in the present invention are notparticularly limited, and they include, for instance, Candida albicansTIMM 1768 strain and Candida albicans ATCC 10231 strain.

In the present specification, the phrase “recognized by antiserum” meansbinding to an antibody component contained in the antiserum, and theantigen can be detected and/or quantified by immunological proceduresused for determining this binding, such as immunoblotting, ELISA, andimmunoprecipitation. The binding of an antigen with an antibody isusually carried out at 4° C. to room temperature in an appropriatesolution or gel, or on an antigen-immobilized plate.

These antigenic proteins are useful in mammals as antigens for vaccinesfor inducing resistance immunity to infection caused by Candida,represented by Candida albicans, and other fungal infections, and asantigens for diagnosing the presence or absence of infection andprogression status thereof. In addition, the antigenic proteins are alsouseful as antigens in the method of prophylaxis, treatment and diagnosisof allergoses caused by fungi, represented by Candida albicans. Inaddition, because Candida albicans is a normally colonizing fungus inhuman bodies, most human immune cells (lymphocytes, macrophages, and thelike) cause immune reactions, such as release of various cytokines andactivation of immune cells in response to the antigenic proteins derivedfrom Candida albicans.

Concretely, the antigenic protein of the present invention is useful asan antigen for releasing or activating effective immunoregulators, suchas interferon γ and interleukin 4. Also, the antigenic protein of thepresent invention can be used as an antigenic component administered toindividuals in, for example, provocation test, skin test, and nasal oreye mucosa test, for the purpose of in vivo diagnosis. Furthermore, theantigenic protein of the present invention can also be used as anantigenic component in laboratory diagnoses, including, for example,diagnostic methods based on agglutination reactions, precipitationreactions, neutralization reactions and labeled antibody techniques,which are antigen-antibody reactions; histamine release test; lymphocyteblast formation test; and leukocyte migration inhibition test.

The antigenic protein of the present invention is not particularlylimited, as long as it is a protein or polypeptide, which are recognizedby antiserum derived from a mammal having Candida albicans-infectionresistance. The above protein or polypeptide can be obtained byimmunoscreening for, for instance, an expression product of cDNA libraryfrom Candida albicans using antiserum derived from a mammal havingCandida albicans-infection resistance.

Incidentally, the term “protein” in the present specification means amajor component of living organisms, and refers to those comprising apolypeptide chain. In addition, the polypeptide in the presentspecification means those resulting from binding of a plurality of aminoacids via peptide bonding, and the number of constituting amino acids isnot particularly limited. In addition, the polypeptide in the presentspecification may be either a simple peptide comprising only aminoacids, or a polypeptide complex comprising components other than theamino acids. Further, the term “fusion protein” is used in the presentspecification, which means a protein or polypeptide resulting frombinding a partial or entire portion each of two or more proteins.

Concrete examples of the antigenic proteins include, for instance, thefollowing polypeptides.

-   -   (A-1) a polypeptide having any one of amino acid sequences as        shown in SEQ ID NOs: 1 to 9;        (A-2) a polypeptide having an amino acid sequence resulting from        deletion, substitution, insertion or addition of one or more        amino acids, for instance, one or several amino acids, in any        one of the amino acid sequences as shown in SEQ ID NOs: 1 to 9;        (A-3) a polypeptide encoded by a nucleic acid having any one of        the nucleotide sequences as shown in SEQ ID NOs: 10 to 18; and        (A-4) a polypeptide encoded by a nucleic acid having a        nucleotide sequence resulting from deletion, substitution,        insertion or addition of one or more bases, for instance, one or        several bases, in any one of the nucleotide sequences as shown        in SEQ ID NOs: 10 to 18.

In addition, a protein immunologically equivalent to the antigenicprotein mentioned above is encompassed in the present invention.

Here, the polypeptides each having the amino acid sequences as shown inSEQ ID NOs: 1 to 9 are each derived from a cDNA library of Candidaalbicans TIMM 1768 strain, obtained by immunoscreening each polypeptideusing antiserum derived from a mammal having Candida albicans-infectionresistance. In addition, in the immunoscreening, when an expressionlibrary of which host is Escherichia coli is used, the polypeptide isexpressed as a simple polypeptide. Therefore, an antigenic protein isscreened with an antibody against a polypeptide moiety by removing theinfluence of an antibody against sugar chains contained at a high levelin anti-Candida serum.

The polypeptide having the amino acid sequence as shown in SEQ ID NO: 1of Sequence Listing consists of 248 amino acids, and has homology totriosephosphate isomerase from Saccharomyces cerevisiae [T. Alber and G.Kawasaki, Journal of Molecular and Applied Genetics, 1, 419-434 (1982)].Since N-terminus methionine is detached according to the results ofN-terminus amino acid analysis of a protein isolated from Candidaalbicans cultured cells, it is considered that a part easily utilizableas an antigenic protein at least exists in a region from 2nd to 248thamino acids in SEQ ID NO: 1 of Sequence Listing. Further, whenconsidered together with the results of homology analysis, thepolypeptide as shown in SEQ ID NO: 1 of Sequence Listing is determinedto be a full length structure of the triosephosphate isomerase ofCandida albicans.

The polypeptide having the amino acid sequence as shown in SEQ ID NO: 2of Sequence Listing consists of 132 amino acids, and has homology to aregion (amino acid NOs: 10 to 137 amino acids) near the N-terminus oflysyl tRNA synthase from Saccharomyces cerevisiae [consisting of 590amino acids, M. Mirande and J. P. Walker, Journal of BiologicalChemistry, 263, 18443-18451 (1988)].

The polypeptide having the amino acid sequence as shown in SEQ ID NO: 3of Sequence Listing consists of 548 amino acids, and a part of a regionfrom 260th to 546th amino acids in the polypeptide has homology to aregion of 523rd to 868th amino acids in SEQ ID NO: 3 of YCR030C onchromosome 3 from Saccharomyces cerevisiae [encoding a proteinconsisting of 870 amino acids; M. R. Red et al., Yeast, 7, 533-538(1991)].

The polypeptide having the amino acid sequence as shown in SEQ ID NO: 4of Sequence Listing consists of 175 amino acids, and has homology toEGD2 from Saccharomyces cerevisiae [consisting of 174 amino acids; M.Johnston et al., Science, 265, 2077-2082 (1994)].

The polypeptide having the amino acid sequence as shown in SEQ ID NO: 5of Sequence Listing consists of 88 amino acids, and has homology to aregion (amino acid NOs: 158 to 242) near the C-terminus of ATP synthasedelta strand from Saccharomyces cerevisiae [consisting of 244 aminoacids; J. Velours et al., European Journal of Biochemistry, 170, 637-642(1988)].

The polypeptide having the amino acid sequence as shown in SEQ ID NO: 6of Sequence Listing consists of 264 amino acids, and has homology toBMH2 from Saccharomyces cerevisiae [consisting of 272 amino acids; G. P.H. von Heusden et al., European Journal of Biochemistry, 229, 45-53(1995)].

The polypeptide having the amino acid sequence as shown in SEQ ID NO: 7of Sequence Listing consists of 224 amino acids, and has homology to aribosomal YL8 protein from Saccharomyces cerevisiae [consisting of 243amino acids; K. Mizuta et al., Nucleic Acids Res., 20, 1011-1016(1992)].

The polypeptide having the amino acid sequence as shown in SEQ ID NO: 8of Sequence Listing consists of 115 amino acids, and a part of from 46thamino acid to the C-terminus in SEQ ID NO: 8 has homology to a part of284th to 353rd amino acids of YNL083w from Saccharomyces cerevisiae[consisting of 494 amino acids; A. Soler-Mira et al., Yeast, 12, 485-491(1996)].

The polypeptide having the amino acid sequence as shown in SEQ ID NO: 9of Sequence Listing is a known HSP70 SSB type polypeptide, which hasbeen found as an antigenic protein recognized by rabbit anti-Candidaserum. Its full length consists of 613 amino acids, and the polypeptideconsisting of 280th to the C-terminus (333 amino acids) of SEQ ID NO: 9is obtained as an antigenic protein [V. Maneu et al., Yeast, 13, 677-681(1997)].

On the other hand, in the present invention, in the cDNA relating to theamino acid sequence as shown in SEQ ID NO: 9 of Sequence Listing, thereare obtained four kinds of cDNAs exhibiting positive results inimmunoscreening of the expression products, wherein each of theseencodes a region of 320th amino acid to the C-terminus (294 aminoacids); a region of 452nd amino acid to the C-terminus (162 aminoacids); a region of 496th amino acid to the C-terminus (118 aminoacids); and a region of 513th amino acid to the C-terminus (101 aminoacids) in the amino acid sequence as shown in SEQ ID NO: 9 of SequenceListing. Therefore, it is thought that a polypeptide of a length of atleast 100 amino acids at the C-terminus is important to show theantigenicity. From the above, polypeptides having about 100 to about 294amino acids containing at least 100 amino acids at the C-terminus,including the above four kinds of proteins, are encompassed in theantigenic protein of the present invention. In other words, in the aminoacid sequence as shown in SEQ ID NO: 9 of Sequence Listing of thepresent invention, a polypeptide resulting from elimination of aminoacids having a length of 319 to 512 amino acids from the N-terminus areencompassed in the antigenic protein in the present invention.

In the present invention, polypeptides having an amino acid sequenceresulting from deletion, substitution, insertion or addition of one ormore amino acids, for instance, one or several amino acids, in each ofthe polypeptides having any one of amino acid sequences as shown in SEQID NOs: 1 to 9 are encompassed in the antigenic protein of the presentinvention, as long as they are recognized by antiserum derived from amammal having Candida albicans-infection resistance. Here, the term“several” refers to a number of several or greater. For instance, theantigenic proteins as shown in SEQ ID NO: 1 and SEQ ID NO: 2 areobtained as a fusion protein resulting from addition of a peptidecomprising T7 tag at the N-terminus, and the protein or polypeptideresulting from addition of a peptide unrelated in the antigenicity asdescribed above is encompassed in the antigenic protein of the presentinvention, and a polypeptide resulting from deletion of the T7 taggedportion is encompassed in the antigenic protein of the presentinvention. In addition, there is cited a polypeptide resulting fromdeletion of about 319 to about 512 amino acids from the N-terminus inthe amino acid sequence as shown in SEQ ID NO: 9 in Sequence Listing asan example of deletion, which is encompassed in the antigenic protein inthe present invention.

In addition, in the present invention, a polypeptide having a part ofthe amino acid sequences as shown in SEQ ID NOs: 1 to 9 of SequenceListing is encompassed in the antigenic protein of the presentinvention, as long as they are recognized by antiserum derived from amammal having Candida albicans-infection resistance. Further,polypeptides having an amino acid sequence resulting from deletion,substitution, insertion or addition of one or more amino acids, forinstance, one or several amino acids, in an amino acid sequence having apart of the amino acid sequences are also encompassed in the antigenicprotein of the present invention, as long as they are recognized byantiserum derived from a mammal having Candida albicans-infectionresistance. In addition, a protein or polypeptide resulting fromaddition of a peptide unrelated in the antigenicity to these antigenicproteins of the present invention is encompassed in the antigenicprotein of the present invention.

The polypeptide having a part of the amino acid sequences shown in SEQID NOs: 1 to 9 of Sequence Listing can be obtained, for instance, byusing an antigenic protein having the amino acid sequences as shown inSEQ ID NOs: 1 to 9 of Sequence Listing as a raw material, cleaving theraw material by enzymatic digestion using a protease such as lysylendopeptidase or trypsin, or cleaving by chemical treatment withcyanogen bromide or the like, and thereafter isolating and purifying adesired peptide fragment having the antigenicity by a known method inthe purification of a protein. Further, the polypeptide can be alsoproduced by chemical synthesis utilizing the peptide synthesis techniqueon the basis of the information on the chemical structure of thepolypeptide obtained by the above method, or the like. In addition, thepolypeptide can be produced by genetic engineering manipulations byusing a nucleic acid encoding an amino acid sequence of the polypeptide.

As the means for carrying out deletion, substitution, insertion oraddition of one or more, for instance, one or several amino acids, in anamino acid sequence, it would not be difficult to carry out such meansby the use of various genetic engineering means described in MolecularCloning: A Laboratory Manual, 2nd Ed. (published by Cold Spring HarborLaboratory in 1989, edited by T. Maniatis et al.), or the like.

Each of the nucleotide sequences as shown in SEQ ID NOs: 10 to 18 ofSequence Listing is an example of a sequence for a nucleic acid encodinga polypeptide consisting of each of the amino acid sequences as shown inSEQ ID NOs: 1 to 9. Therefore, in the present invention, a protein orpolypeptide recognized by antiserum derived from a mammal having Candidaalbicans-infection resistance, the polypeptide encoding a nucleic acidconsisting of each of the nucleotide sequences as shown in SEQ ID NOs:10 to 18 of Sequence Listing is encompassed in the antigenic protein ofthe present invention.

The nucleic acid is cDNA capable of expressing an antigenic proteinobtained by immunoscreening of the expression library, wherein the cDNAis a full length of the gene, or has a partial nucleotide sequence. Forinstance, the nucleotide sequences as shown in SEQ ID NO: 10 and SEQ IDNO: 15 of Sequence Listing are full lengths of the antigenic proteingenes, and the nucleotide sequences as shown in SEQ ID NO: 11, SEQ IDNO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, andSEQ ID NO: 18 of Sequence Listing are partial sequences of the antigenicprotein genes. For instance, when an antigenic protein derived fromHSP70 SSB-type is the smallest antigenic protein of above, for instance,a polypeptide consisting of 101 amino acids from 513th to the C-terminusof the amino acid sequence as shown in SEQ ID NO: 9 of Sequence Listing,the antigenic protein encoded by the nucleic acid as shown in SEQ ID NO:18 is encoded by the nucleotide sequence of 580th to 882nd bases of thenucleotide sequence of SEQ ID NO: 10. Therefore, it is preferable that arecombinant DNA encoding a portion having high antigenicity is designedto include 580th to 882nd bases of SEQ ID NO: 10 of the nucleotidesequence of Sequence Listing.

In addition, in the present invention, a polypeptide encoded by anucleic acid having a nucleotide sequence resulting from deletion,substitution, insertion or addition of one or more bases, for instance,one or several bases, in each of the nucleotide sequences as shown inSEQ ID NOs: 10 to 18 of Sequence Listing is encompassed in the antigenicprotein of the present invention, as long as the polypeptide isrecognized by antiserum derived from a mammal having Candidaalbicans-infection resistance. Here, the term “several” refers to anumber of several or greater.

In order to carry out deletion, substitution, insertion or addition ofone or more bases, for instance, one or several bases, in each of thenucleotide sequences as described above, there may be utilized variousknown genetic engineering manipulations, including, for instance, gappedduplex method [Wilfried, K. et al., Nucleic Acids Research, 12,9441-9456 (1984)], deletion method [Celeste, Y. P. et al., Methods inEnzymology, 154, 367-382 (1987)], uracil DNA method [Thomas, A. K. etal., Gene, 34, 315-323 (1987)], cassette mutation method [James, A. W.et al., Gene, 34, 315-323 (1985)], and the like.

In addition, a protein derived from Candida albicans mutant, a closelyrelated species (for instance, fungi belonging to the genus Candida),and fungi other than Saccharomyces cerevisiae, the protein orpolypeptide having characteristics which are immunologically equivalentto the antigenic protein of the present invention is encompassed in theantigenic protein of the present invention. The term “protein havingcharacteristics which are immunologically equivalent” in the presentspecification means a protein or polypeptide which is recognized by anantibody contained in antiserum derived from a mammal immunized by theantigenic protein of the present invention having any one of the aminoacid sequences as shown in SEQ ID NOs: 1 to 9 and has differentmodification in the amino acid sequence or sugar chain.

Incidentally, the antigenic protein of the present invention may bemodified to give a derivative, for the purpose of enhancement ofinduction of individual prophylactic immunocompetent, the decrement ofallergic reaction or disappearance of enzyme activity, when used inenhancement of stability and/or enhancement of a desired reactivity asan antigen, namely when used in treatment; or for the purpose ofenhancement of specific binding of an antigen with an antibody, whenused for diagnosis. The modification method includes, for instance,pyridylethylation, reduction, alkylation, acylation, chemical couplingto an appropriate material, mild formalin treatment or guanidinehydrochloride treatment. As the method for preparing the derivative, forinstance, by using polyethylene glycol (PEG) method [Wie et al., Int.Arch. Allergy Appl. Immunol., 64, 84-99 (1981)], the antigenic proteinmay be allowed to bind with PEG.

2. Nucleic Acid of Present Invention

The nucleic acid of the present invention is a nucleic acid encoding anantigenic protein which is recognized by antiserum derived from a mammalhaving Candida albicans-infection resistance.

The above nucleic acid can be obtained, for instance, in the same manneras the antigenic protein of the present invention, by preparing cDNAexpression library from Candida albicans, and immunoscreening theexpression library by using antiserum derived from a mammal havingCandida albicans-infection resistance. In addition, the nucleic acid canbe also obtained by preparing a genomic library or cDNA library ofCandida albicans, and screening of the library by using anoligonucleotide as a probe, the oligonucleotide being deduced to encodea part of the amino acid sequence of the antigenic protein of thepresent invention.

The nucleic acid of the present invention is useful as an antigen genefor vaccines for inducing resistance immunity against infections causedby Candida, represented by Candida albicans, and other fungalinfections; and as an antigen gene for prophylaxis, treatment anddiagnosis of allergoses caused by fungi, represented by Candidaalbicans.

Concrete examples of the nucleic acid of the present invention, forinstance, include the following nucleic acids:

-   (B-1) a nucleic acid encoding a polypeptide having any one of amino    acid sequences as shown in SEQ ID NOs: 1 to 9;    -   (B-2) a nucleic acid encoding a polypeptide having an amino acid        sequence resulting from deletion, substitution, insertion or        addition of one or more amino acids, for instance, one or        several amino acids, in any one of the amino acid sequences as        shown in SEQ ID NOs: 1 to 9;    -   (B-3) a nucleic acid having any one of nucleotide sequences as        shown in SEQ ID NOs: 10 to 18;    -   (B-4) a nucleic acid having an nucleotide sequence resulting        from deletion, substitution, insertion or addition of one or        more bases, for instance, one or several bases, in any one of        nucleotide sequences as shown in SEQ ID NOs: 10 to 18; and-   (B-5) a nucleic acid capable of hybridizing with a nucleic acid    according to any one of (B-1) to (B-4), or a nucleic acid    complementary thereto, under stringent conditions.

Here, the nucleic acid may be either DNA or RNA.

Each of the nucleotide sequences shown in SEQ ID NOs: 10 to 18 ofSequence Listing is an example of a sequence of a nucleic acid encodinga polypeptide having the amino acid sequences as shown in SEQ ID NOs: 1to 9.

Incidentally, there has been known that 1 to 6 codons (triplet basecombination) designating a particular amino acid on a gene exist forevery amino acid. Therefore, there can be a large number of kinds ofnucleic acids each encoding an amino acid sequence, depending on itsamino acid sequence. In nature, the nucleic acid does not exist in astable form, and it is not rare that a mutation of its nucleotidesequence takes place. The mutation on the nucleic acid may not affectthe amino acid sequence encoded thereby (so-called “silent mutation”),in which case the nucleic acid having the above mutation can be said asdifferent nucleic acids encoding the same amino acid sequence. Therecannot, therefore, be denied the possibility that even when the nucleicacid encoding a particular amino acid sequence is isolated, a variety ofnucleic acids encoding the same amino acid sequence are produced withgeneration passage of the organism containing them. Moreover, it is notdifficult to artificially produce a variety of the nucleic acidsencoding the same amino acid sequence by means of various geneticengineering manipulations.

For example, when a codon used on a natural DNA encoding the desiredprotein is low in usage in the host in the production of a protein bygenetic engineering, the expression level of the protein is sometimeslow. In such a case, high expression of the desired protein is achievedby artificially converting the codon into another one of commonly usedin the host without changing the amino acid sequence encoded (forexample, Japanese Examined Patent Publication No. Hei 7-102146). It isof course possible to artificially produce a variety of nucleic acidsencoding a particular amino acid sequence, and the nucleic acids can bealso produced in nature.

From the above, the nucleic acid encoding a polypeptide having each ofthe amino acid sequences as shown in SEQ ID NOs: 1 to 9, which are givenin (B-1) in the present specification is not limited to the nucleic acidhaving each of the nucleotide sequences as shown in SEQ ID NOs: 10 to18, which are given in (B-3).

Therefore, in the present invention, a nucleic acid having a nucleotidesequence resulting from deletion, substitution, insertion or addition ofone or more bases, for instance, one or several bases in each of thenucleotide sequences as shown in SEQ ID NOs: 10 to 18 of SequenceListing is also encompassed in the nucleic acid of the presentinvention, as long as its expression product is recognized by antiserumderived from a mammal having Candida albicans-infection resistance.Here, the term “several” refers to a number of several or greater.

For instance, there are also encompassed in the nucleic acid of thepresent invention a nucleic acid, which is a derivative of a nucleicacid as shown in SEQ ID NOs: 10 to 18 of Sequence Listing, the nucleicacid encoding a protein resulting from appropriate detachment of a partof the protein with retaining the antigenicity of each of the antigenicprotein having the amino acid sequences as shown in SEQ ID NOs: 1 to 9of Sequence Listing, or a nucleic acid encoding a fusion protein of theantigenic protein in which another polypeptide, for instance, a proteinderived from T7 phage, histidine tag, maltose binding protein,glutathione-S-transferase, β-galactosidase, or the like is added.

Further, a nucleic acid capable of hybridizing with a nucleic acid asshown in any one of (B-1) to (B-4), or with a nucleic acid complementarythereto, under stringent conditions, the nucleic acid encoding anantigenic protein which is recognized by antiserum derived from a mammalhaving Candida albicans-infection resistance [i.e. the nucleic acid of(B-5)] is also encompassed in the nucleic acid of the present invention.The above nucleic acid can be detected as follows.

In other words, a membrane immobilized with the nucleic acid to bedetected is incubated at 50° C. for 12 to 20 hours together with a probein 6×SSC (wherein 1×SSC shows 0.15 M NaCl, 0.015 M sodium citrate, pH7.0) containing 0.5% by weight SDS, 0.1% by weight bovine serum albumin(BSA), 0.1% by weight polyvinyl pyrrolidone, 0.1% by weight of asynthetic, water soluble, nonionic copolymer of sucrose andepichlorhydrin (FICOLL® 400), and 0.01% by weight denatured salmon spermDNA. After termination of the incubation, the membrane is washed,initiating under the conditions of 37° C. in 2×SSC containing 0.5% byweight SDS, the SSC concentration being made variable up to a range of0.1×SDS, and the temperature being variable up to a range of 50° C.,until a signal ascribed to an immobilized nucleic acid can bedistinguished from the background, and thereafter the probe is detected.When the nucleic acid is detected under the conditions described above,the nucleic acid is referred to “a nucleic acid capable of hybridizingunder stringent conditions.” The probe used herein is a part of anucleic acid as shown in any one of (B-1) to (B-4), or a part of anucleic acid complementary thereto.

The nucleic acid described above includes, for instance, a nucleic acidcapable of hybridizing with a nucleic acid as shown in any one of (B-1)to (B-4), or a nucleic acid complementary thereto, under stringentconditions, possessed by mutants and closely related fungi of Candidaalbicans (for instance, the fungi of the genus Candida such as Candidatropicalis), the nucleic acid encoding a protein having immunologicallyequivalent antigenicity.

In addition, in the nucleic acid of the present invention, a nucleicacid accompanied by its mutant form, allele, homologue gene, degeneracyof codon in a nucleic acid encoding a polypeptide having any one of theamino acid sequences as shown in SEQ ID NOs: 1 to 9, or a nucleic acidencoding a polypeptide having an amino acid sequence resulting formdeletion, substitution, insertion or addition of one or more aminoacids, for instance, one or several amino acids, in any one of the aminoacid sequence as shown in SEQ ID NOs: 1 to 9 is encompassed in thenucleic acid of the present invention, as long as its expression productis recognized by antiserum derived from a mammal having Candidaalbicans-infection resistance.

Further, the nucleic acid of the present invention includes a nucleicacid encoding an antigenic protein recognized by antiserum derived froma mammal having Candida albicans-infection resistance, for instance, anucleic acid specifically binding to the nucleic acids shown in (B-1) to(B-5). Here, the nucleic acid of the present invention may be labeledwith a known fluorescent substance or radioactive substance. The nucleicacid can be utilized for a probe used for detection of an antigenicprotein gene of Candida albicans by hybridization, or for a primer in amethod for amplifying DNA using the DNA polymerase.

3. Vector and Transformant of Present Invention

The vector of the present invention is a vector comprising the nucleicacid of the present invention. Such a vector enables a host such asEscherichia coli to stably harbor the nucleic acid of the presentinvention. The vector usable for construction of the above vectorincludes, for instance, pUC118, pWH5, pTV118, PSCREEN-1B, and the like,and the vector is not particularly limited as long as it is a vector inwhich the nucleic acid of the present invention can be inserted andexpressed. In addition, the DNA of the present invention can be used byligating to an appropriate expression vector, to give recombinant DNAfor expression. In the present invention, each of the nucleic acidshaving the nucleotide sequences as shown in SEQ ID NOs: 10 to 18 is usedby ligating to PSCREEN-1B, and thereafter introducing the resultingproduct as recombinant DNA for expression into Escherichia coli. Themethod for inserting a nucleic acid into a vector is not particularlylimited, and there can be employed a known method, such as a methodusing T4 DNA ligase.

The transformant of the present invention is those resulting fromtransformation with the vector of the present invention. Thetransformant described above is obtained by introducing the vector ofthe present invention into a host. As the host, there can be used insectcells and animal cells such as COS-7 and Vero cells, in addition tobacteria such as Escherichia coli, the genus Bacillus and the like,yeasts such as Saccharomyces cerevisiae, fungi such as the genusAspergillus, Pichia pastoris. The method for introducing a vector into ahost include known methods, for instance, calcium phosphate method,CaCl₂ method, DEAE dextran method, electroporation method, and the like.The above transformant can be selected by, for instance, using as anindex a selection marker in the used vector. In addition, there can beconfirmed that a desired nucleic acid is introduced by, for instance,hybridization using a nucleic acid extracted from the transformant and aprobe capable of specifically detecting the nucleic acid of the presentinvention.

The transformant described above can be utilized when the nucleic acidof the present invention is prepared and when the antigenic protein ofthe present invention is prepared.

3. Method for Producing Antigenic Protein of Present Invention

The method for producing an antigenic protein which is recognized byantiserum derived from a mammal having Candida albicans-infectionresistance of the present invention is characterized in that the methodcomprises culturing the transformant of the present invention underconditions capable of expressing an antigenic protein encoded by thenucleic acid of the present invention.

The culture conditions are not particularly limited. For instance, in acase of a transformant obtained by using lambda phage vector λSCREEN™-1and Phage Maker™ System Phage Pack Extract [both manufactured byNovagen], the transformant may be cultured in LB medium containingampicillin utilized as a selective pressure, and induced-expression byIPTG (isopropyl-thio-β-D-galactoside) in an appropriate timing. In atransformant obtained by using other vectors and hosts, the transformantmay be also cultured under appropriate conditions capable of expressingproteins in the medium to which an appropriate selective pressure isapplied. The desired protein is expressed by culturing in the mannerdescribed above.

The expressed protein or polypeptide can be collected and purified by aknown protein purification method in the field of art. In addition, thepresence or absence of the antigenicity of the resulting protein orpolypeptide can be confirmed by immunological techniques, for instance,immunoblotting method, ELISA, immunoprecipitation method, and the like.

In addition, the antigenic protein of the present invention may beproduced from the transformant of the present invention obtained bygenetic engineering manipulations, or the antigenic protein may beobtained from Candida albicans culture by a known protein purificationmethod.

4. Pharmaceutical Composition and Diagnostic Composition of PresentInvention

The pharmaceutical composition and diagnostic composition of the presentinvention are 1) those comprising the antigenic protein of the presentinvention, or an antigenic protein obtained by the method of the presentinvention, and 2) those comprising the nucleic acid of the presentinvention.

1) Pharmaceutical Composition and Diagnostic Composition Each ComprisingAntigenic Protein of Present Invention, or Antigenic Protein Obtained byMethod of Present Invention.

In the pharmaceutical composition and diagnostic composition of thepresent invention, there are encompassed a vaccine for inducing inmammals resistance immunity to infection caused by Candida, representedby Candida albicans, and other fungal infection, and a diagnosticcomposition for diagnosing the presence or absence of infection andprogression status thereof. Further, compositions used for prophylaxis,treatment and diagnosis of allergoses caused by fungi, represented byCandida albicans are also encompassed. In addition, because Candidaalbicans are normally colonized in human bodies, most human immune cells(lymphocytes, macrophages, and the like) cause immune reactions, such asrelease of various cytokines and activation of immune cells, to theantigenic proteins derived from Candida albicans. In other words, in thepharmaceutical composition and diagnostic composition of the presentinvention, there are also encompassed compositions for releasing oractivating effective immunoregulators, such as interferon γ andinterleukin 4.

The composition for inducing the resistance immunity to infection isgenerally employed as a preparation in the form of a suspension orsolution of the antigenic protein of the present invention containingthe following adjuvant, in order to acquire further enhanced humoralimmunity and/or cellular immunity. The adjuvant is usually administeredtogether with the antigenic ingredients, and the adjuvant may beadministered before or after administration of the antigenicingredients.

The adjuvant appropriate for vaccination to mammals includes complete orincomplete Freund's adjuvant; inorganic gels made of aluminum hydroxide,alum and the like; detergents such as lysolecithin anddimethyloctadecylammonium bromide; polyanions such as dextran sulfateand poly-IC; peptides such as muramyl peptide and tuftsin; monophophoryllipid A (MPL) manufactured by Ribi; and B subunit of cholera toxin,without being limited thereto. The antigen can be administered byincorporating it in liposome or other microcarriers. There can be ofcourse used a mixture of several different antigenic proteins.

The composition for prophylaxis, treatment and diagnosis of allergosesmay be used in a form of an appropriate salt solution or suspension ofthe antigenic protein of the present invention. In some cases,polyethylene glycol and phenol may be added thereto. Further, thecomposition may be a suspension or solution containing the adjuvantdescribed above. The adjuvant is usually administered together with theantigen, and the adjuvant may be administered before or afteradministration of the antigen. The antigen can be administered byincorporating it in liposome or other microcarriers.

The pharmaceutical composition of the present invention may beformulated with various additives as occasion demands, and its form canhave various preparation forms. The above additives include additivesfor preparation for the purpose of forming a desired preparation form.Examples of these additives include, for instance, nutrients such asascorbic acid, biotin, calcium pantothenate and niacin; covering agentssuch as sodium metaphosphate, sodium phosphates (primary, secondary,tertiary salts) and sodium pyrophosphate; preservatives such as calciumsorbate and benzoic acid; other additives and diluents such as Arabicrubber, traganth, sodium alginate, mannitol, sorbitol, lactose,fructose, soluble starches, amino acids, glucose, sucrose, honey, andfatty acid esters.

The pharmaceutical composition of the present invention can beadministered orally or non-orally. For instance, the pharmaceuticalcomposition may be made into a preparation form suitable for oraladministrations such as powder, granule, pellet, tablets, coatingagents, capsules, solutions and syrup; and preparation forms suitablefor non-oral administrations such as injection, drops, suppository,ophthalmic solutions, collunarium and spray.

When the pharmaceutical composition of the present invention isparticularly used as vaccines, there may be added as occasion demands astabilizer such as human serum albumin, gelatin and amino acids in anappropriate concentration, and a preservative such as phenol andthimerosal in an appropriate concentration. In addition, thepharmaceutical composition may be used as a solution preparation, or adry preparation by lyophilizing the composition. The dry preparation maybe suspended with an appropriate solvent upon use such as distilledwater for injection.

As a method for administering the pharmaceutical composition, thepharmaceutical composition may be administered orally, transmucosally(nasally, intravaginally or the like), percutaneously (subcutaneously orintracutagenously), or intravenously. Representative dosage ispreferably in a range of 0.01 to 5.0 mg/kg body weight as an amount ofprotein, and more preferably in a range of 1 μg to 100 μg/kg bodyweight. As occasion demands, the dosage may be increased, or the numberof dosage may be increased.

The diagnostic composition used against individuals for the purpose ofin vivo diagnosis in, for instance, provocation test, skin test, ornasal or eye mucosa test includes those in which the antigenic proteinof the present invention is made into a form of lyophilized powder or ina form of an appropriate salt solution or suspension, and polyethyleneglycol or phenol may be added to such compositions. For patch tests, theantigenic protein of the present invention may be mixed with whitepetrolatum as a base material supplemented with a detergent such assodium lauryl sulfate. In addition, in a case of using as an antigen forassaying IgE titer, the above antigenic protein can be used byimmobilizing the antigenic protein on a solid material such as a paperdisc, a cellulose sponge, or a microplate.

2) Pharmaceutical Composition and Diagnostic Composition ComprisingNucleic Acid of Present Invention

The pharmaceutical composition and diagnostic composition of the presentinvention can be used in mammals as vaccines for inducing resistanceimmunity to infection caused by Candida, represented by Candidaalbicans, and other fungal infection, and can be used for prophylaxis,treatment and diagnosis of allergoses caused by fungi, represented byCandida albicans. When the composition is used for the protection toinfection and the treatment of allergoses, a plasmid carrying thenucleic acid of the present invention at downstream of an appropriatepromoter may be directly used, or may be prepared in the form of anappropriate salt solution or suspension after its incorporation intoretrovirus or adenovirus, such that the nucleic acid of the presentinvention is capable of expressing in mammalian cells. In addition, thenucleic acid of the present invention may be incorporated in theliposome or other microcarriers, or it may be mixed with an appropriatepolycationic lipid.

As the above nucleic acid, there may be employed those includingchemical modifications which allow to enhance the migration ability intothe cells or the stability within the cells. The chemical modificationsinclude, for instance, derivatives derived from phosphorothioate,phosphorodithioate, alkyl phospho triesters, alkyl phosphonates, alkylphosphamidates and the like.

As a method for administering the pharmaceutical composition, thepharmaceutical composition may be administered intramuscularly,subcutaneously, intravenously, orally, intrarectally, percutaneously,nasally, hypoglosally, intraperitoneally, or the like. Representativedosage is preferably in a range of 0.01 μg to 10 mg/kg body weight as anamount of the nucleic acid, and more preferably in a range of 1 μg to 5mg/kg body weight. As occasion demands, the dosage may be increased, orthe number of dosage may be increased.

5. Antibody or Antibody Fragment of Present Invention

The antibody of the present invention is an antibody capable ofspecifically binding to the antigenic protein of the present invention.Here, as the antibody, an antibody fragment is also encompassed, as longas the antibody fragment is capable of specifically binding to theantigenic protein of the present invention.

The antibody of the present invention can be obtained by a conventionalmethod, and it may be either a polyclonal antibody or monoclonalantibody. In addition, in the present invention, the antibody may be asingle-chain antibody capable of specifically binding to the antigenicprotein of the present invention. The antibody or the antibody fragmentcan be utilized for detection of fungi of the infectious diseases ofCandida albicans or closely related fungi, or it can be utilized foridentification of allergens in allergoses caused by Candida albicans orclosely related fungi.

When the antibody or antibody fragment of the present invention is usedfor detection of fungi of infectious diseases, identification ofallergens in allergoses, and the like, the nucleic acid may be labeledwith a known fluorescent substance or radioactive substance.

In addition, the antibody can be used for purification of the antigenicprotein of the present invention. In this case, for instance, theantigenic protein of the present invention can be more easily purifiedby immobilizing the antibody or antibody fragment of the presentinvention to a carrier or the like.

6. Nucleic Acid Capable of Specifically Binding to Nucleic Acid EncodingAntigenic Protein of Present Invention

The nucleic acid capable of specifically binding to a nucleic acidencoding the antigenic protein of the present invention can be utilizedfor a probe used in detection of a nucleic acid encoding the antigenicprotein of the present invention by hybridization, or it can be utilizedfor a primer used for detection by a method for amplifying DNA using theDNA polymerase. The nucleic acid capable of specifically binding to anucleic acid encoding the antigenic protein of the present inventionincludes those binding to sense side or antisense side of the nucleicacid encoding the antigenic protein. The nucleic acid may be labeledwith a known fluorescent substance or radioactive substance.

EXAMPLES

The present invention is hereinafter described in more concretely bymeans of the following examples, without intending to limit the presentinvention to these examples.

Example 1 Induction of Infection Resistance and Involvement ofCD4-Positive T-Cells in Infection Resistance

1) Induction of Infection Resistance

Candida albicans (C. albicans) TIMM 1768 was cultured overnight withshaking in Sabouraud dextrose medium. Thereafter, cultured cells wereharvested by centrifugation and washed with physiological saline. Theobtained cells were suspended in physiological saline so as to haveconcentrations of 1×10⁶, 1×10⁷, and 1×10⁸ cells/ml. An equal volume ofIFA (incomplete Freund's adjuvant) was added to each suspension andmixed. This mixture was subcutaneously administered to C57BL/6 mice at0.1 ml per animal, thereby immunizing the mice with live Candida cells.After 1 week, the mice were further immunized by subcutaneouslyadministering the same number of live Candida cells prepared in the samemanner as above. Therefore, each mouse was twice immunized with 5×10⁴,5×10⁵, or 5×10⁶ live Candida cells. As a control, a mixture ofphysiological saline and an equal volume of IFA, the physiologicalsaline being used in place of a suspension of live Candida cells, wastwice administered subcutaneously at an interval of 1 week.

One week after the second immunization, all immunized mice and controlmice were infected by intravenous administration of 2.5×10⁵ C. albicansTIMM 1768 cells obtained by culturing in Sabouraud-dextrose medium.After infection, the mice were observed for life or death for 30 days.The results are shown in Table 1. TABLE 1 Number of Live Mean Mice after30 Dosage at Survival ± days/Number of Administration One Time SD DaysMice Used Physioogical —  4.0 ± 1.4 0/5 Saline Live Cells 5 × 10⁴ 16.8 ±6.3 0/5 5 × 10⁵ 19.6 ± 9.0 0/5 5 × 10⁶ >20.8 ± 10.1 2/5

Table 1 shows that all control mice (non-immunized mice) died within 10days at the dose levels mentioned above, whereas the immunized miceacquired obvious resistance to infection.

2) Acquisition of Infection Resistance by CD4-Positive T Cells

An anti-CD4 antibody (prepared from ATCC TIB207) or anti-CD8 antibody(prepared from ATCC TIB105) was intraperitoneally administered to BALB/cmice that had been twice immunized by 5×10⁶ live Candida albicans cellsat an interval of 1 week in the same manner as in item 1) above, at 0.3mg per animal 3 times in total at 23, 26, and 29 days after the finalimmunization. At 29 days after final immunization, 1×10⁵ C. albicansTIMM 0136 cells obtained by culturing in Sabouraud-dextrose medium wereintravenously administered. As a control, non-immunized mice, and miceimmunized without administering any antibody, were prepared, and thesemice also were intravenously administered with 1×10⁵ C. albicans TIMM0136 cells in the same manner as above, respectively. At 7 days afteradministration, the number of live C. albicans cells in the kidney wasdetermined.

The anti-CD4 antibody and anti-CD8 antibody were purified by aconventional method from ascitic fluid in which hybridomas wereproliferated by injecting hybridomas intraperitoneally to scid mice. Inaddition, by using a flow cytometer (manufactured by Ortho Diagnostic),it was confirmed that the obtained antibody could eliminate respectivecells.

As a result, it is concluded that the decrease of the Candida cellnumber in the kidney is significantly inhibited by the administration ofthe anti-CD4 antibody, indicating that CD4-positive T-cells play animportant role in the infection resistance.

Example 2 Collection of Serum from Mammal Having Infection Resistance,Characteristics Thereof, and Preparation of Various Absorption Sera

1) Collection of Serum from Infection-Resistant Mammal

Serum was collected from BALB/c mice that were immunized 3 times with5×10⁶ live C. albicans TIMM 1768 cells in a mixture with CFA (completeFreund's adjuvant) in the same manner as in item 2) of Example 1 by aconventional method at 7 days after final immunization. This resultingserum was used as an anti-Candida serum.

2) Characteristics of Serum from Infection-Resistant Mammal

The characteristics of the anti-Candida serum obtained in item 1) ofExample 2 were studied.

First, as antigenic components, C. albicans cell wall fraction (CW),cytoplasm fraction (HSS), and cell membrane fraction (LSP) wereprepared.

A loopful of C. albicans TIMM 1768 cells in Sabouraud agar slant culturewas transferred to a test tube containing 5 ml of YPD medium (1% byweight yeast extract, 2% by weight polypeptone, 2% by weight glucose).After culturing with shaking at 30° C. for 24 hours, 50 μl of theresulting culture was inoculated to 500 ml of YPD medium contained in a2-liter conical flask and cultured overnight with shaking at 35° C. Atotal of four such 2-liter conical flasks were used to culture.

Cells were harvested by centrifugation at 2,000×g for 10 minutes fromabout 2 liters of the culture obtained (about 7×10⁷ cells/ml). The cellswere washed twice with 500 ml of sterilized water, and then washed oncewith 500 ml of the SSB solution (50 mM phosphate buffer, pH 7.5,containing 0.8 M sorbitol). After the cells were suspended in about 500ml of the SSB solution, 70 ml of SSB solution containing 100 mM EDTA,and 1 ml of 2-mercaptoethanol were added thereto, and then gentlyshaken. Subsequently, 70 ml of SSB solution containing 3.3 mg ofZYMOLYASE® 20T (manufactured by Seikagaku Corporation) per 1 ml wasadded to the resulting suspension, and then gently shaken at 35° C. for1 hour. Further, 70 ml of SSB solution containing 12 mg of TrichodermaLysing Enzyme (manufactured by Sigma) per 1 ml was added, and thengently shaken at 35° C. for 1 hour. The suspension obtained wascentrifuged at 2,000×g for 10 minutes to harvest protoplast cells, whilethe supernatant was taken as the cell wall fraction (CW).

One-hundred and forty milliliters of sterile physiological saline wasadded to the protoplast cells obtained in the manner described above(the protoplast cells contained per 1 ml of this suspension being about1×10⁹ cells), and then stirred well. Thereafter, the mixture was allowedto stand on ice for 10 minutes. After confirming that the protoplastcells bursted, the mixture was centrifuged at 10,000×g for 30 minutes,and the precipitate obtained was taken as an insoluble fraction(referred to as LSP). The centrifugal supernatant was furthercentrifuged at 100,000×g for 60 minutes, and the supernatant obtainedwas taken as the soluble fraction (referred to as HSS). After suspendingthe LSP in 140 ml of physiological saline, the resulting suspension wassubjected to sonication treatment, and then sterilized by heat treatmentin a boiling water bath for 5 minutes, to give an LSP antigen solutioncontaining membrane proteins and the like. In addition, the CW was alsosubjected to sonication and heat treatments in the same manner as above,to give a CW antigen solution. As to the HSS, it was used without anytreatment as an HSS antigen solution.

The protein concentrations of the LSP antigen solution, the HSS antigensolution, and the CW antigen solution obtained in the manner asdescribed above were 2.3 mg/ml, 3.5 mg/ml, and 2.1 mg/ml, respectively(the amount of protein was quantified by using bicinchonic acid (BCA)reagent with BSA as a standard).

Each of the CW antigen solution, the LSP antigen solution, and the HSSantigen solution obtained as antigenic components was diluted with PBSso as to have a protein concentration of 10 μg/ml. Thereafter, 50 μl ofeach dilution was added to IMMUNO-MODULE (manufactured by Nunc), andeach of the IMMUNO-MODULE was allowed to stand at 4° C. overnight tocoat them with the respective antigens. The coated IMMUNO-MODULE wassubjected to blocking treatment by using PBS solution containing 1% byweight bovine serum albumin. Next, 50 μl of a 60-fold dilutedanti-Candida serum or control serum was added to each IMMUNO-MODULE. Themixture was incubated at 37° C. for 1 hour, and thereafter incubatedwith a peroxidase-labeled anti-mouse IgG antibody (2,000-fold diluted)at 37° C. for 1 hour. After incubation, the mixture was washed with PBS,and a substrate solution was then added thereto. After 15 minutes, theabsorbance at 405 nm was determined. The higher the absorbance shows thelarger the amount of antigen-recognizing antibody, i.e. the higher theantibody titer against the antigen. The results are shown in Table 2.The above control serum was prepared from serum derived from a mouseadministered with a mixture of physiological saline and an equal volumeof CFA, the physiological saline being used in place of live cells, in 3times subcutaneously at intervals of 1 week. TABLE 2 Antigen AbsorbanceFraction Anti-Candida Serum Control Serum CW 0.013 0.006 LSP 0.051 0.006HSS 0.237 0.006

As is evident from Table 2, there was elucidated that the anti-Candidaserum has the highest antibody titer against the HSS but possesses anantibody titer against the LSP as well. The antibody titer against theCW was low.

Example 3 Screening of C. albicans cDNA Expression Library for AntigenicProteins

1) Preparation of C. albicans cDNA Expression Library

Total RNA was extracted and purified from cells to prepare a cDNAexpression library for C. albicans TIMM 1768 strain. Specifically, theabove strain was cultured at 35° C. in 200 ml of YPD medium, andthereafter the cells were harvested by centrifugation (2,000 rpm, 5minutes) and washed once with distilled water. The cells were quicklyfrozen with liquid nitrogen, and the frozen cells were disrupted with amortar into a powdery form. Total RNA was recovered and purified fromthis cell powder using an RNA extraction kit (manufactured byPharmacia). poly(A)⁺ RNA was prepared using Oligotex™-dT30 <Super>(manufactured by Takara Shuzo Co., Ltd.) from this total RNA. cDNA wassynthesized from 5 μg of the poly(A)⁺ RNA using cDNA synthesis kit(manufactured by Takara Shuzo Co., Ltd.). A cDNA library was constructedby ligating the synthesized cDNA to lambda phage vector λSCREEN™-1(manufactured by Novagen), and thereafter carrying out in vitropackaging by using Phage Maker™ System PhagePack Extract (manufacturedby Novagen).

2) Immunoscreening of Antigenic Proteins Recognized by the Anti-CandidaSera Resulting from Immunization with Live C. albicans Cells

Phage clones expressing proteins reactive with the antisera resultingfrom immunization with live C. albicans cells were detected byimmunoscreening. In the cDNA library prepared by phage vectorλSCREEN™-1, the cDNA was expressed as a fusion protein with a peptidecomprising the T7 tagged sequence etc., or a polypeptide initiating fromthe translation initiation codon in the cDNA.

Specifically, the cDNA library was inoculated to host Escherichia coliBL21(DE3)pLysE strain and mixed with top agarose (LB medium containing0.7% by weight agarose). Thereafter, the mixture was overlayered on aplate of 2×YT (1.6% by weight Bacto-tripton, 1% by weight yeast extract,0.5% by weight NaCl) and cultured at 37° C. for 6 hours, to form phageplaques. A nylon membrane (Hybond™-N, manufactured by Amersham) wassuperposed on the resulting plaque, and thereafter allowed to standovernight at 4° C. and subsequently incubated at 37° C. for 4 hours. Themembrane was removed from the plate, and then washed with PBST(composition: 100 mM NaCl, 10 mM phosphate buffer, pH 7.5, 0.1% Tween®20) for 10 minutes, and thereafter blocked by immersing the membrane inan appropriate amount of BLOCK ACE (manufactured by DainipponPharmaceutical). This membrane was allowed to stand at room temperatureovernight, and thereafter incubated with the anti-Candida serum preparedin item 1) of Example 2 (500-fold dilution) at room temperature for 3hours. After incubation, the membrane was washed, and thereafterincubated with a peroxidase-labeled anti-mouse IgG antibody (1,000-folddilution) at room temperature for 1 hour. After incubation, the membranewas washed, and then allowed to generate luminescence by usingSuperSignal® Substrate Western Blotting (manufactured by Pierce),thereby detecting phage plaques reactive to the anti-Candida serum.

The nucleotide sequences of the above novel genes, i.e., the S.cerevisiae triosephosphate isomerase homologue gene, the S. cerevisiaelysyl-tRNA synthetase homologue gene, and the homologue gene to DNA 98on chromosome 3 of S. cerevisiae, are respectively as shown in SEQ IDNO: 10, SEQ ID NO: 11, and SEQ ID NO: 12 in Sequence Listing. From theabove nucleotide sequences, these nucleic acids encode the polypeptidescomprising the amino acid sequences respectively as shown in SEQ ID NO:1, SEQ ID NO: 2, and SEQ ID NO: 3 in Sequence Listing.

3) Screening for C. albicans Antigenic Proteins Using AbsorptionAnti-Candida Serum

The cDNA library described in item 1) above was immunoscreened for C.albicans antigenic proteins using an absorption antiserum from the serumimmunized with live C. albicans cells. Specifically, a solution in whichthe anti-Candida serum, the HSS, and the CW were mixed, in a 1:1:1(v/v/v) ratio, was prepared and used as an absorption antiserum.

Antigenic molecules were screened in the same manner as in item 2) ofExample 3 using this absorption antiserum. The antiserum used was a500-fold dilution of the absorption antiserum. Phage plaques reactivewith this antiserum were further cloned. As a result of the nucleotidesequence analysis of the cloned cDNA, it was clarified that one clonecomprises a nearly full length of a novel gene having homology to the S.cerevisiae EGD2 gene, and the other clone is a part of a novel genehaving homology to the ATP synthase delta chain. The obtained nucleotidesequence information is respectively shown in SEQ ID NO: 13 and SEQ IDNO: 14 in Sequence Listing. In addition, each of these nucleotidesequences encodes the polypeptide having the amino acid sequence shownin SEQ ID NO: 4 or SEQ ID NO: 5 in Sequence Listing.

Also, four clones comprising a part of a known cDNA of the HSP70 SSBtype were obtained, each being cDNA encoding 294 amino acids, 162 aminoacids, 118 amino acids, and 101 amino acids of the C-terminus side,respectively. As a result, it was clarified that the HSP70 SSB typeantigen has an antigenic determinant reactive to the antiserum withinthe region consisting of 118 amino acid of the C-terminus side. Thenucleotide sequence of the HSP70 SSB type gene encoding the longest 294amino acids, and the amino acid sequence deduced therefrom are as shownin SEQ ID NO: 18 and SEQ ID NO: 9, respectively.

Next, there existed phage plaques which were positive against theabsorption antiserum obtained by addition of HSS but negative againstthe above absorption antiserum obtained by addition of the HSS and theCW. The cDNAs of these plaques were analyzed in the same manner as initem 2) above. Two clones comprised a full length of a novel gene havinghomology to the S. cerevisiae BMH 2 gene. One clone comprised a nearlyfull length of another novel gene having homology to the S. cerevisiaeribosomal protein L7 gene. Another one clone contained a part (115 aminoacids) of a novel gene having homology to S. cerevisiae YNL 083W.

The nucleotide sequences of these novel genes, i.e., the S. cerevisiaeBMH 2 homologue gene, the S. cerevisiae ribosomal L7 protein homologuegene, and the S. cerevisiae YNL 083W homologue gene, are respectivelyshown in SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17 in SequenceListing. From the above nucleotide sequences, each of these genesencodes the polypeptide having the amino acid sequences respectivelyshown in SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8 in SequenceListing.

Example 4 Production of Antigenic Proteins which were Recognized byAntisera from Mammals Having C. albicans Infection Resistance

The cDNAs obtained in items 2) and 3) of Example 3, except the genehaving homology to S. cerevisiae YNL 083W, expressed genes as fusionproteins. The gene having homology to YNL 083W expressed a polypeptidefrom the initiation codon (ATG) in the cDNA. As an example of theexpression of a fusion protein, polypeptides expressed from a genehaving homology to triosephosphate isomerase (TPI) and from a genehaving homology to lysyl-tRNA synthase were clarified.

Specifically, a plasmid carrying the gene having homology to TPI (triosephosphate isomerase) obtained in item 2) of Example 3, and a plasmidcarrying the gene having homology to lysyl-tRNA synthase, were eachtransformed into Escherichia coli BL21(DE3)pLysS to give transformants.Each of these transformants was inoculated to LB medium containingampicillin, and cultured at 37° C. for 3.5 hours. IPTG(isopropyl-1-thio-β-D-galactoside) was added to the resulting culture soas to have a final concentration of 1 mM, and thereafter the culture wascontinued for additional 2.5 hours. The culture was centrifuged toharvest the cells, and the cells were washed and suspended in 30 μl of1×SDS sample buffer. The suspension was subjected to heat treatment at100° C. for 5 minutes, and thereafter the suspension was centrifuged. Apart of the supernatant was subjected to SDS-polyacrylamide gel (12.5%gel) electrophoresis, thereby confirming the expression of apolypeptide.

The deduced molecular weights of the expressed fusion proteins are 63kDa (27 kDa for TPI (triose phosphate isomerase) homologue+36 kDa for T7tagged region) for the TPI (triose phosphate isomerase) homologue, and50 kDa (14 kDa for lysyl-tRNA synthetase homologue+36 kDa for T7 taggedregion) for the lysyl-tRNA synthetase homologue. FIG. 1 clearly showedthat these fusion proteins are expressed as polypeptides having therespective deduced molecular weights.

The sample of lane 1 in FIG. 1 is a polypeptide obtained before theinduction of expression of the transformant in which the lysyl-tRNAsynthase homologue gene (before addition of IPTG) was transduced; andthe sample of lane 2 is a polypeptide obtained after the induction(after addition of IPTG). The sample of lane 3 comprises the polypeptideobtained before the induction of expression of the transformant in whichthe TPI homologue gene was transduced, and the sample of lane 4 is thepolypeptide obtained after the induction.

INDUSTRIAL APPLICABILITY

The antigenic protein of the present invention is an antigenic proteinwhich is recognized by antiserum derived from a mammal having Candidaalbicans-infection resistance, so that the antigenic protein is usefulfor treatment and diagnosis of Candida albicans-infection. The nucleicacid of the present invention is that encoding the antigenic protein ofthe present invention, so that the nucleic acid is useful for treatmentand diagnosis of Candida albicans-infection.

1. An isolated antigenic polypeptide characterized in that the antigenicprotein is recognized by antiserum from a mammal having Candida albicansinfection resistance, wherein said antigenic polypeptide is: (A-1) apolypeptide having any one of amino acid sequences as shown in SEQ IDNOs.: 2, 4, 6 and 7; (A-2) a polypeptide encoded by a nucleic acidhaving any one of the nucleotide sequences as shown in SEQ ID NOs.: 11,13, 15 and 16; and (A-3) a polypeptide encoded by a nucleic acid capableof hybridizing with a nucleic acid according to any one of having anyone of (A-1) and (A-2), or complementary thereto, under stringentconditions.
 2. An isolated nucleic acid encoding an antigenicpolypeptide according to claim
 1. 3. A method for producing an antigenicpolypeptide which is recognized by antiserum from a mammal havingCandida albicans infection resistance, characterized in that the methodcomprises culturing the transformant resulting from transformation withthe vector which comprises the isolated nucleic acid according to claim2.
 4. A pharmaceutical composition characterized in that thepharmaceutical composition comprises the antigenic polypeptide accordingto claim 1 or the nucleic acid according to claim
 2. 5. A diagnosticcomposition characterized in that the diagnostic composition comprisesthe antigenic polypeptide according to claim
 1. 6. A diagnosticcomposition characterized in that the diagnostic composition comprisesthe nucleic acid according to claim
 2. 7. An antibody capable ofspecifically binding to the antigenic polypeptide according to claim 1.8. A nucleic acid capable of specifically binding to the nucleic acidaccording to claim 2.